Gastroenterology

Gastroenterology

Volume 125, Issue 3, September 2003, Pages 854-867
Gastroenterology

Basic-liver, pancreas, and biliary tract
Bid activates multiple mitochondrial apoptotic mechanisms in primary hepatocytes after death receptor engagement

https://doi.org/10.1016/S0016-5085(03)01066-7Get rights and content

Abstract

Background & Aims: Activation of Fas or tumor necrosis factor receptor 1 (TNF-R1) on hepatocytes leads to apoptosis, which requires mitochondria activation. The pro-death Bcl-2 family protein, Bid, mediates this pathway by inducing mitochondrial releases of cytochrome c and other apoptotic factors. How Bid activates mitochondria has been studied in vitro with isolated mitochondria. We intended to study the mechanisms in intact hepatocytes so that findings could be made in a proper cellular context and would be more physiologically relevant. Methods: Hepatocytes were isolated from wild-type and bid-deficient mice and treated with anti-Fas or TNF-α. Mechanisms of mitochondria activation were dissected with genetic, biochemical, and morphologic approaches. Results:bid-deficient hepatocytes were much more resistant to apoptosis. Bid was required for permeability transition and mitochondria depolarization in addition to the previously defined release of cytochrome c. Permeability transition inhibitors cyclosporin A and aristolochic acid could inhibit mitochondria activation effectively, but not as much as the deletion of the bid gene, and they could not inhibit Bak oligomerization. In addition, mitochondria depolarization also could be induced by caspases, whose activation was mainly dependent on Bid. Conclusions: Bid may activate mitochondria by 2 mechanisms, one is related to permeability transition and the other is related to Bak oligomerization. Bid can further affect mitochondria potentials by indirectly regulating caspase activity. This in vivo study provides novel findings not previously disclosed by in vitro studies, and indicates the importance of several mechanisms in contributing Bid-mediated mitochondria dysfunction that could be potential cellular targets of intervention.

Section snippets

Mice

Mice deficient in bid were established by gene targeting and had been back-crossed to the C57BL/6 background for 12 generations.12, 13 Wild-type and bid-deficient mice used in this study were littermates and weighed about 25–30 g. They were maintained as homozygotes in a specific pathogen-free facility on the campus in compliance with the National Institutes of Health and University of Pittsburgh policies.

Establishment of primary hepatocyte culture

Murine hepatocytes were isolated using the collagenase digestion method as previously

Hepatocytes deficient in bid are refractory to anti-Fas or TNF-α

To study the mechanism by which Bid activates the mitochondria in the cell, we had set up a primary hepatocyte culture and verified the role of Bid in anti-Fas- or TNF-α—induced apoptosis in this system. Cell viability was monitored at different times after the treatment by MTT assay (Figure 1A), nuclear staining (Figure 1B), trypan blue staining, and visual inspection by phase microscopy (data not shown). Data from all these assays were comparable and showed that a majority of wild-type

Discussion

A number of studies on death receptor—induced hepatocyte apoptosis have been conducted, but some of the key questions remained unanswered, particularly those about mitochondria activation. Because the analysis can be performed in a proper cellular environment, the cell culture system provides a unique way to examine these issues, which may not be readily addressable by the animal studies or the in vitro reconstitution analysis. We thus aimed to determine the mechanism of mitochondria activation

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  • Cited by (0)

    Supported in part by the Howard Temin Award (K01 CA 74885, National Institutes of Health), R01 CA 83817, National Institutes of Health (to X.-M.Y.), and P01 DK59340, National Institutes of Health (to J.J.L.).

    1

    Y.Z. and W.-X.D. contributed equally to this work.

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